The proposed project will develop fast and reliable mass spectrometry procedures for quantitative detection of cellular protein complexes involved in HIV-1 transcription regulation and associated with protein phosphatase-1 and Tat protein. In addition we will study changes in phosphorylation of these proteins upon the use of PP1-targeted inhibitors of HIV-1. In Specific Aim 1 we will develop MS based methodologies for quantitative protein analysis. We will focus on increase of MS sensitivity and on protein sequence coverage improvement. In Specific Aim 2 we will apply the quantitative MS methods to the analysis of PP-1 complexes involved in HIV-1 regulation. Specifically, we will analyze PP1-associated protein complexes that target PP1 to CDK9, Sp1 or RNAPII and other yet unknown proteins. In Specific Aim 3, we will study the effect of PP1-targeted small molecule HIV-1 inhibitors on PP1-associated protein complexes, their interaction and phosphorylation. A detailed understanding of the HIV-1 proteome and phosphoproteome will be used for future drug design, targeting HIV-1 through the inhibition of PP1. A successful accomplishment of the project will improve understanding of HIV-1 mechanisms, will identify potential HIV-1 drug targets, and will give detailed information about the role of PP1 regulation in HIV-1 inhibition. This project will also stimulate refinement and development of new quantitative protocols for proteomics and phospho-proteomics. PUBLIC HEALTH RELEVANCE: In the proposed study we will develop mass spectrometry methodology and apply it to the analysis of protein phosphatase-1 complexes involved in HIV-1 regulation. We will also analyze the effect of PP1-targeted small molecule HIV-1 inhibitors using quantitative MS approaches. A successful accomplishment of the project will improves HIV-1 mechanism understanding, will identify potential HIV-1 drug targets, will give detailed information about HIV-1 PP1 regulation and inhibition. This project will also stimulate refinement and development of new quantitative protocols for proteomics and phospho-proteomics.